EBNA3C is critical for IRF4-mediated cell proliferation by inhibiting apoptosis.

<p>10 million HEK-293 were transfected with Ctrl-vector and different combinations of expression plasmids for Myc-tagged EBNA3C, Flag-tagged IRF4 and N-terminal domain deleted mutant Myc-tagged EBNA3C (residues 366–992). In addition, cells were transfected with GFP expression vector. Transfected cells were selected for 2 weeks with G418 antibiotic. A) After 2 weeks of selection, GFP fluorescence of each plate was scanned by a PhosphorImager and the area of the colonies calculated by Image J software. C) The numbers of colonies in different transfected sets are represented in bar diagram. The data represented in bar diagram is the average of three independent experiments. B) 0.1×10⁶ cells from each set of selected samples were plated and cultured for 6 days. Viable cells were counted at indicated time points by trypan blue dye exclusion technique. D) G418 selected stable cells were harvested, lysed in RIPA buffer and subjected to immunoblot analyses with indicated antibodies. E, F, H) Sh-Ctrl, Sh-IRF4 LCL1 cells were grown in RPMI medium for 12 hrs with or without etoposide. E) Cells were harvested and stained with Propidium iodide. Stained cells were subjected to Flowcytometric analysis. G) The bar diagram represents the fold change of apoptosis seen by cell cycle analysis using FACS. The results shown are representative of three independent experiments. F) 1×10⁶ cells were plated and allowed them to grow at 37°C in complete medium without puromycin antibiotic. Viable cells were counted by trypan blue dye exclusion technique at indicated time points. The results shown here are representative of three independent experiments. H) Cells were treated with increasing amount (0, 10, 20, 40, 80, 160 µM) of etoposide drug for 12 hrs. Cell lysates were used for Western blot analysis with PARP-1, GAPDH antibodies.